The purpose of the proposed research is to characterize the biochemical pathway of conversion of n-hexadecane to palmitate by Acinetobacter calcoaceticus. The approach to this problem will involve both physiological and genetic studies. Mutants which are unable to grow on hexadecane will be isolated using transpositional mutagenesis. Insertion mutants will be classified into genetically similar groups based on genetic transformation and will be characterized phenotypically by their ability to grow on several putative intermediates of hexadecane oxidation. Insertion mutants from each genetic and phenotypic class will be examined for the accumulation of intermediates resulting from the partial oxidation of hexadecane. Products resulting from the oxidation of radioactive labeled hexadecane will be identified using TLC, GLC, and GLC-mass spectrometry. Genetic analysis will be performed using transformation and conjugation. Insertion mutants will be mapped against each other and in relation to auxotrophic mutations. Insertion mutations will be localized on large regions of the chromosome and map order and linkage determined for any clustered alk loci. This study will: 1) define the biochemical pathway of hexadecane oxidation in Acinetobacter, 2) define genetic loci involved in the pathway, and 3) result in a preliminary map of genes necessary for hexadecane oxidation.